Antibodies are immunological proteins that bind a specific antigen. In most mammals, including humans and mice, antibodies are constructed from paired heavy and light polypeptide chains. Each chain is made up of two distinct regions, referred to as the variable (Fv) and constant (Fc) regions. The light and heavy chain Fv regions contain the antigen binding determinants of the molecule and are responsible for binding the target antigen. The Fc regions define the class (or isotype) of antibody (IgG for example) and are responsible for binding a number of Fc receptors and other Fc ligands, imparting an array of important functional capabilities referred to as effector functions. Several key features of antibodies including but not limited to, specificity for target, ability to mediate immune effector mechanisms, and long half-life in serum, make antibodies powerful therapeutics. Recombinant screening methods for isolating antibodies with a desired binding specificity have been developed. For example, it is possible to generate large expression libraries of binding molecules using combinatorial recombinant DNA technologies. This is especially true in the field of antibody engineering, where recombinant antibody libraries routinely contain more then 109 unique clones. The availability of large libraries of binding molecules has provided a source of binders to most any ligand.
Surface display libraries allow for the enrichment of specific binding clones by subjecting the organism displaying the binding molecule (e.g., phage and yeast) to successive rounds of selection (e.g., panning; for reviews see, Trends Biotechnol 9: 408-414; Coomber, et al., 2002, Methods Mol Biol 178: 133-45, Kretzschmar et al., 2002, Curr Opin Biotechol 13: 598-602; Fernandez-Gacio, et al., 2003, Bioorg Med Chem Lett. 13:213-216; Lee et al., 2003, Trends Biotechnol 21: 45-52; and Kondo, et al., 2004, Appl Microbiol Biotechnol 64: 28-40). In particular, advances in phage display antibody libraries have made them an attractive alternative to screening conventional hybridoma-derived monoclonal antibodies. Phage display library screening is advantageous over some other screening methods due to the vast number of different polypeptides (typically exceeding 109) that can be contained in a single phage display library. This allows for the screening of a highly diverse library in a single screening step.
Display of small peptides or single chain proteins on phage can be advantageous as long as intracellular processing or post-translational modification (of which phage or prokaryotic hosts are not capable) is not necessary or desired. For example, effective display of a heterologous polypeptide may require various post-translational modifications, intracellular structures, and a compliment of specialized enzymes and chaperone proteins that are necessary to transport, to glycosylate, to conform, to assemble, and to anchor the display polypeptide properly on the surface of the host cell; however, none of these processes can be accomplished by bacteriophage or prokaryotic cell processes. Furthermore, prokaryotic cells do not always efficiently express functional eukaryotic proteins.
Bacterial and bacteriophage display systems are also limited by the small capacity of the display system, and as such, are more suited for the display of small peptides as are the recently developed methods for surface display of small peptides on mammalian cells (see, e.g., Wolkowicz, et al., 2005, J. Biol. Chem., 280: 15195-15201). As a result bacteriophage and mammalian antibody display libraries and methods require that only a fragment of an antibody be displayed on the surface. If the purpose is to discover “whole” antibodies then the antibody fragments must be cloned into a whole antibody. Not only does this add an extra step, but also many antibody fragments have decreased affinity for an antigen when converted to a whole antibody and such libraries. Furthermore, such methods cannot be used to examine the binding of other antibody domains such as the Fc region to antibody receptors (e.g., Fc receptors) or other Fc ligands.
Whole antibody cell surface display systems have been developed for some eukaryotic cells, such as yeast (see, e.g., Boder and Wittrup, 2000, Methods in Enzymology, 328:430-444), but the develop of whole antibody display on mammalian cells lags behind. Furthermore, the size of the libraries, which can be generated in these systems, is limited. Since the chance of isolating antibodies with the desired binding properties from an antibody library is proportional to the size and diversity of the library there is a need for methods to generate large and diverse libraries. This is particularly important if you want to build a naive antibody library for antibody discovery, for example from un-immunized donors. Currently, to build the library size larger than 108 members is a challenge to any eukaryotic cell display technology by using conventional transfection tools such as transient transfection or electroporation. Thus, there is a need for antibody cell surface display libraries and library screening methods for eukaryotic cells, in particular mammalian cells, which maintain a large diversity, but eliminate any of the issues discussed supra. Such a system would be particularly useful for the identification of antibody variants in regions outside of the variable domain such as in the Fc region. Modifications, including amino acid deletions, substitutions and additions, of the Fc region have been demonstrated to alter the binding of the Fc region to its ligands and/or receptors resulting in a concomitant change in effector function (see, e.g., (Shields et al., 2001, J Biol Chem 276:6591-6604 and Presta et al., 2002, Biochem Soc Trans 30:487-490 and U.S. Patent Publication 2004/0132101). Thus, by modifying the Fc region the therapeutic effectiveness of Fc containing molecules can be improved. A system for whole antibody cell surface display on mammalian cells would facilitate the rapid identification of antibodies with modified Fc regions having altered effector function.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.